Genetically engineered vaccine of epsilon toxin of clostridium perfringens and application thereof

A Clostridium perfringens, genetic engineering vaccine technology, applied in the direction of genetic engineering, application, gene therapy, etc., can solve the problems of economic loss in animal husbandry, fatal intestinal diseases, etc., achieve non-toxic side effects, avoid vaccine concentration , the effect of strong immunity

Inactive Publication Date: 2014-06-04
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The epsilon toxin is produced by Clostridium perfringens type B and type D, which can cause fatal intestinal diseases in goats, sheep and other animals, causing great economic losses to animal husbandry

Method used

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  • Genetically engineered vaccine of epsilon toxin of clostridium perfringens and application thereof
  • Genetically engineered vaccine of epsilon toxin of clostridium perfringens and application thereof
  • Genetically engineered vaccine of epsilon toxin of clostridium perfringens and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Expression and purification of Clostridium perfringens epsilon toxin protein

[0041] 1. Design primers

[0042] According to the ε toxin gene sequence submitted on Genebank (accession number: M95206, its sequence is shown in Seq ID No: 1), amplify the ε toxin part-specific gene fragment, design primers: primer1 and primer2, and add Ecor1 and Xho1 respectively Restriction sites;

[0043] primer1: 5'-ACTGAATTCAAGGAAATATCTAATACAGTA-3'Ecor1, its sequence is shown in Seq ID No:3

[0044] primer2: 5'-ACTCTCGAGTTATTTTTATTCCTGGTGCCTA-3'Xho1, its sequence is shown in Seq ID No:4

[0045] 2. Prepare the template

[0046] Use an inoculating loop to pick the D-type Clostridium perfringens strain and inoculate it into a blood plate medium (100 ml of distilled water, 1 g of glucose and 3.8 g of bean agar powder) after sterilization, add 5 ml of sheep blood and divide it into six cells. blood plate), 37°C anaerobic (88% N 2 , 7%H 2 , 5%CO 2 ) cultured for 36h. Take a...

Embodiment 2

[0095] Example 2: Expression of epsilon toxin protein and determination of its toxicity

[0096] Using the absorption value of CS29000 thin-layer gel scanner (Shanghai Kehe Biochemical Technology Co., Ltd.) at a wavelength of 560 nm, it was determined that the expressed product accounted for 32.53% of the total bacterial protein.

[0097] The culture supernatant (ie, supernatant 1) of the recombinant strain BL21(DE3)-ε, the bacterial cell (recombinant strain BL21(DE3)-ε) and the bacterial cell lysate (the bacterial liquid after complete lysis in step 5) and the production Clostridium perfringens virulent D8346 culture supernatant was respectively inoculated on egg yolk agar plate (weighed 50g of finished agar medium, added 1L of deionized water, stirred and heated to boil until completely dissolved, divided into conical flasks, sterilized at 121 ℃ for 15min, Cool to about 50℃, add 10-15mL of 50% sterile yolk saline per 100mL of medium, shake well and pour into six plates immed...

Embodiment 3

[0099] Example 3: Safety inspection of epsilon toxin genetically engineered vaccine

[0100] (1) 3 guinea pigs weighing 250g-350g were injected subcutaneously with 5 ml of the vaccine (can be injected into two parts), observed for ten days, and all were healthy.

[0101] (2) 4 rabbits weighing 1.5-2 kg were injected intramuscularly with 5 ml of the vaccine and observed for 10 days. All were healthy and alive, and no necrosis occurred at the injection site.

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Abstract

The invention discloses a genetically engineered vaccine of epsilon toxin of clostridium perfringens and an application thereof. The genetically engineered vaccine of epsilon toxin of clostridium perfringens is obtained by the following steps: firstly, designing a specific primer; carrying out PCR (Polymerase Chain Reaction) amplification to obtain an epsilon gene segment, and cloning to a pMD18-T Vector; selecting a positive recombinant, and guiding a recombinant plasmid into an acceptor strain to successfully construct a recombinant strain BL21 (DE3)-epsilon; and finally, emulsifying the prepared epsilon-toxin protein and an aluminum hydroxide adjuvant in a volume ratio of 9:1, and adding 0.01% of thiomersalate to obtain the genetically engineered vaccine of epsilon toxin of clostridium perfringens. The genetically engineered vaccine of epsilon toxin of clostridium perfringens can be put into industrial production easily, simple to operate, has good safety and the like, and can be applied to preventing fetal intestinal diseases of animal such as lambs, sheep, goats, calves and chinchilla, caused by D type clostridium perfringens.

Description

(1) Technical field [0001] The invention relates to a Clostridium perfringens epsilon toxin genetic engineering vaccine and application thereof, belonging to the technical field of animal genetic engineering. (2) Background of the invention [0002] Clostridium perfringens (Clostridium perfringens), also known as Clostridium welchii, is an opportunistic pathogen widely distributed in nature and in the intestinal tract of animals. According to the different types of exotoxins (mainly α, β, ε, ι) produced by bacteria, Clostridium perfringens can be divided into five types: A, B, C, D, and E. The ε toxin is produced by Clostridium perfringens type B and type D, which can cause fatal intestinal diseases in goats, sheep and other animals, and cause great economic losses to animal husbandry. [0003] The coding gene etx of Clostridium perfringens ε toxin is located on the plasmid, and the protein ε toxin precursor encoded by etx gene is avirulent in the early stage of secretion, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/08A61K9/107C12N15/31C12N15/70C07K14/33A61P31/04
Inventor 柴同杰王叶
Owner SHANDONG AGRICULTURAL UNIVERSITY
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